The goal of this project is to understand how the C-terminal domain of RNA polymerase II is used to couple transcription with several post-initiation steps in gene expression. These events include mRNA capping, splicing, and polyadenylation, as well as regulation of transcription elongation and termination. Current data supports a model in which the pattern of CTD phosphorylation changes at different stages of transcription. Each phosphorylation state may be recognized by a distinct set of CTD-interacting proteins. This allows a dynamic exchange of elongation and mRNA processing factors, each one recruited at the appropriate time(s) of the transcription cycle. The experiments in this project will test this model and identify physical and functional relationships between the CTD, its various kinases and phosphatases, and its associated elongation and mRNA processing factors. Five specific aims are proposed. The first aim will use chromatin immunoprecipitation to survey the crosslinking patterns of all known elongation and mRNA processing factors. Once the wild-type patterns are known, the experiments will be repeated in various mutant strains. Mutants to be assayed include CTD kinases and phosphatases, as well as the elongation and processing factors themselves. Changes in patterns will suggest an interdependence for association with transcription complexes. In the second aim, affinity chromatography will used to isolate proteins that bind to specific phosphorylated forms of the CTD. Proteins identified will be further characterized as to their roles in gene expression. Specific Aim 3 will be to develop in vitro systems for reproducing some of the CTD modification changes observed in vivo. These in vitro systems will be used to test and extend the models of factor interactions derived from Aims 1 and 2. Specific Aim 4 will be to identify and characterize a putative CTD serine 5 phosphatase. Specific Aim 5 will be to decipher the role of the Bur1/Bur2 kinase complex in regulating events occurring during transcription elongation. The experiments proposed would significantly extend our understanding of how various steps in gene expression are integrated. It is clear that post-transcription initiation events are regulated in many systems for modulation of gene activity. A clear understanding of the fundamental mechanisms of gene expression will provide the groundwork for future therapies, including gene replacement therapies and direct modulation of cellular and viral gene expression.